Changes in the strain composition of DWV complexes in honeybee pupae following direct injection of virus.

Levels of the DWV- and VDV-1 CP-coding RNA determined by qRT-PCR (left panel) in the virus preparations used for injection, and (right panel) in pupae following incubation for 3 days. (A) Left panel: ΔCt values for the DWV-type and VDV-1-type CP were obtained by subtracting Ct values for the corresponding CP from Ct for the total DWV-like viruses quantified using “Universal” primers to the NS gene. Right panel: Ct values for the DWV-type and VDV-1-type CP. Six pupae were used for each virus-injected group, three pupae were used for each of the buffer-injected and non-injected control groups. Bars show mean value with standard error. Letters above the bars represent statistically significant groupings according to pairwise t-test comparisons for VDV-1 CP (p-value <0.01). (B) High-throughput sequencing of the virus preparations from the honeybees of group C (left), and the virus accumulated in the pupae injected with 20 ng of the virus preparation (right), 3 days post injection. The graphs show depth of coverage at genomic loci in DWV (red) and VDV-1 (blue) determined by high-throughput sequencing of viral RNA aligning to the DWV and VDV-1 sequences (GenBank Accession numbers GU109335 and AY251269 respectively). Only reads unambiguously aligning to DWV or VDV-1 sequences were used, with up to 3 mismatches tolerated in the 18 nt. seed region. The percentages of DWV, VDV-1 and the DWV-VDV-1 recombinants predicted by MosaicSolver [40] are shown below. The pileup graphs for DWV and VDV-1 are shown, respectively, in red and dark blue. The CP-coding region of the virus C preparation and the virus C-injected pupae, which shows a decrease of DWV coverage compared to the injected virus, is highlighted in yellow.